The determination of the pKa of histidine residues in proteins by Raman difference spectroscopy

Sensitive Raman difference spectroscopy was used to monitor the protonation and deprotonation of histidine residues in apo-transferrin. We have shown previously that the behavior of small molecules and/or small molecular groups bound to proteins or other large macromolecules can be studied by Raman difference spectroscopy (Yue, K.T. et al. (1989) J. Raman Spectrosc. 20, 541-545). Using this method, we have measured the Raman difference spectra of human transferrin at different pH values with respect to pH 8.9, titrating its various histidine residues. About 12 +/- 2 of the 19 residues were titrated. The pH difference spectrum of transferrin obtained is very similar to that of histidine in solution, but with clear differences in the 1200-1400 cm-1 region. A titration curve with pKa of 6.08 +/- 0.01 fit the data of histidine in solution and a value of 6.56 +/- 0.02 was found for the average value of the 12 histidine residues inside transferrin. The technique has enough sensitivity at present to monitor a single histidine residue in a 130 kDa molecule and to determine the titration curve of one residue in a 40 kDa protein.     

Functional analysis of Arg-308 mutants of Flp recombinase. Possible role of Arg-308 in coupling substrate binding to catalysis

The arginine residue at position 308 in the Flp recombinase corresponds to the only invariant arginine within the Int family of recombinases. Alterations of this residue result in Flp variants that retain substrate recognition, but form weaker protein-DNA complexes than wild type Flp. Furthermore, their DNA cleavage activity is significantly diminished. A conservative change of R308K results in a functional Flp variant; however, this protein has a lowered temperature optimum for recombination. The Arg-308 mutants can be stabilized on the DNA substrate through cooperativity with a partner Flp mutant that is tight binding. Thus, interactions between Flp monomers must be a relevant feature of the normal recombination reaction.     

The seminiferous growth factor induces proliferation of TM4 cells in serum-free medium

The seminiferous growth factor (SGF) of the mammalian testes induces DNA synthesis and cell proliferation of Balb/c 3T3 cells (Bellvé and Feig, 1984; Rec Prog Hormone Res 40:531-567). In this study, SGF was purified 80,000- to 100,000-fold from calf testes and used to examine the growth of TM4 cells in a chemically defined medium. Cells were seeded sparsely in Dulbecco's Modified Eagles/Ham's F12 medium (1:1;v:v) (DME/F12 degrees), containing epidermal growth factor (EGF; 1 ng/ml), insulin (1; 10 micrograms/ml), and transferrin (Tr; 5 micrograms/ml) (DME/F12). After 24 h, the medium was replaced with DME/F12 degrees supplemented with SGF, EGF, 1, or Tr, in two-, three- or four-way combinations. Cell numbers were quantified after another 48 h of culture. EGF, I, and Tr, alone or in two-way combinations, were not mitogenic for TM4 cells. By contrast, SGF (1 U) alone, or with any two of these factors, stimulated TM4 cell proliferation to commensurate levels, and to twofold greater numbers than occurred with the combination of EGF, I, and Tr. Synergisms or inhibitions were not measurable. Follicle-stimulating hormone, luteinizing hormone, prolactin, acidic fibroblast growth factor, or basic fibroblast growth factor was weakly or not mitogenic for TM4 cells. The effect of SGF on cell proliferation was inhibited by 1 microM - 1 nM retinoic acid, but not by retinol or retinyl acetate. SGF was mitogenic for bovine adrenal capillary endothelial cells, an effect that was potentiated by 10 micrograms heparin/ml. Thus, SGF can induce proliferation of TM4 cells and capillary endothelial cells. The former provides a sensitive, and selective, serum-free, bioassay system for SGF activity.     

2000-2020年青海湖流域植被降水利用效率时空变化

植被降水利用效率(PUE)是评价植被生产力对降水量时空动态响应特征的重要指标。以年净初级生产力(NPP)数据、年降水量数据为基础,利用地理信息系统(GIS)和遥感(RS)技术,计算并研究了2000-2020年青海湖流域植被降水利用效率时空分布格局及其地形效应,结合年均气温、年均地表温湿度、年生长季光合有效辐射吸收系数和年植被覆盖度等数据,探讨了PUE与各因子间的相关关系。结果表明:(1)青海湖流域单位像元(1 km²)PUE平均值在0.4-0.7 gC m^-2 mm^-1间变化,平均为0.54 gC m^-2 mm^-1,且在年际间无显著变化趋势(R²=0.05, P≥0.05)。在空间上,青海湖流域多年PUE平均值环湖呈现不均匀分布,除青海湖东岸外,PUE值随湖面距离增大呈减小趋势;其高值区主要集中分布在青海湖西岸和南岸的半环区;年PUE变化趋势的斜率值为-0.05-0.04 gC m^-2 mm^-1 a^-1,其中显著变化的区域占流域面积的29.63%。(2)青海湖流域多年PUE平均值在海拔效应和坡度坡向两种不同微地形效应下表现出明显的差异。海拔每升高50 m,PUE值将减少0.02 gC m^-2 mm^-1;随坡度增加,PUE值呈降低趋势,平坡至险坡(>45°)的变化范围为0.3-0.61 gC m^-2 mm^-1;不同坡向PUE值表现为由东北坡向西南坡递减,范围为0.52-0.56 gC m^-2 mm^-1。(3)在空间上,青海湖流域PUE值与地表温度、光合有效辐射吸收系数、植被覆盖度和叶面积指数相关性较为明显。沿海拔梯度,空气温度和地表温度与PUE呈极显著正相关(R²=0.94, P<0.01; R²=0.98, P<0.01),光合有效辐射吸收系数、植被覆盖度和叶面积指数与PUE显著正相关(R²=0.89, P<0.05; R²=0.90, P<0.05; R²=0.86, P<0.05),地表土壤湿度与PUE无显著相关性(R²=0.16, P≥0.05)。评估了青海湖流域植被降水利用效率的特征及其与各因子间的相关关系,明确了植被对降水的利用能力及其耗水特性,可为青海湖流域植被保护和国家公园建设提供理论参考。     

Nontraditional translation is the key to UFMylation and beyond

The Ubiquitin-fold modifier 1 (Ufm1) is a ubiquitin-like protein that can also be conjugated to protein substrates and subsequently alter their fates. Both UFMylation and de-UFMylation are mediated by Ufm1-specific proteases (UFSPs). In humans, it is widely believed that UFSP2 is the only active Ufm1 protease involved in Ufm1 maturation and de-UFMylation, whereas UFSP1 is thought to be inactive. Here, Liang et al. provide strong evidence showing that human UFSP1 is also an active Ufm1 protease. These results solve an age-old mystery in the human Ufm1 conjugation system and could have a greater impact not only on Ufm1 biology but also on the translation of genes employing nontraditional start codons.     

The nascent polypeptide-associated complex (NAC) controls translation initiation in cis by recruiting nucleolin to the encoding mRNA

Protein aggregates and abnormal proteins are toxic and associated with neurodegenerative diseases. There are several mechanisms to help cells get rid of aggregates but little is known on how cells prevent aggregate-prone proteins from being synthesised. The EBNA1 of the Epstein-Barr virus (EBV) evades the immune system by suppressing its own mRNA translation initiation in order to minimize the production of antigenic peptides for the major histocompatibility (MHC) class I pathway. Here we show that the emerging peptide of the disordered glycine–alanine repeat (GAr) within EBNA1 dislodges the nascent polypeptide-associated complex (NAC) from the ribosome. This results in the recruitment of nucleolin to the GAr-encoding mRNA and suppression of mRNA translation initiation in cis. Suppressing NAC alpha (NACA) expression prevents nucleolin from binding to the GAr mRNA and overcomes GAr-mediated translation inhibition. Taken together, these observations suggest that EBNA1 exploits a nascent protein quality control pathway to regulate its own rate of synthesis that is based on sensing the nascent GAr peptide by NAC followed by the recruitment of nucleolin to the GAr-encoding RNA sequence.